CS59492A3 - Canglemycin c, process of its preparation, its use, a pharmaceutical and astrain for said canglemycin c preparation - Google Patents

Description

Kanglemycin C, the method of composition and strain for k

Technique of the present invention use, pharmaceuticals L ^ njyginu 6 CJ 1! i_ Zm> o <-c mN, 1

• JO • o

The present invention relates to kanglemycin C which is a physiologically active substance, a process for its preparation and its use as a pharmaceutical agent. More particularly, the invention relates to kanglemycin C, which is used as a novel immunosuppressant for the treatment of autoimmune hemolytic anemia, renal inflammation and connective tissue rejection, and a method of preparing kangemycin G and pharmaceutical use.

Background Art

Recently developed immunosuppressants are highly selectively effective in the specific regulation of a subset of certain immune cells. Cyclosporin A, which represents such immunosuppressants, selectively inhibits T cell clone helper proliferation and achieves remarkable results in the field of organ transplantation. Cyclosporin A (Agents and Actions, 1976, Vol. 6,468-475) has been extensively used in clinics. However, cyclosporin is accompanied by side effects such as acute (or chronic) renal intoxication, mild tremor, neural lesions, gum disease, hair loss, etc. FK-506 (Journal of Antibiotics, Vol. 40, 1249 (1987) )) which has recently been discovered, the antibiotic macrolide (44) is very promising at experimental level. However, due to its complicated process, it is likely that clinical application of this drug may be limited.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a more convenient immunosuppressant that can overcome the drawbacks of conventional immunosuppressants.

It is a second object of the present invention to provide a process for the preparation of a immunosuppressant which is advantageous in industrial production.

It is a further object of the present invention to provide a microorganism used to prepare a novel immunosuppressant. 2

It is a further object of the invention to provide a pharmaceutical use of a novel immunosuppressant.

The inventors have worked extensively on studies aimed at obtaining a novel immunosuppressant in which the non-advantages of known immunosuppressants are eliminated. As a result of the work of kanglemycin C, it has an extremely small relative molecular weight, which is a novel immunosuppressant. Kanglemycin C was obtained by fermentation of a nutrient broth belonging to the genus Nocardia, which was changed to Amycolatopsis (International Journal of Osteopathic Bacteology, Jan. 1986, p. 29-37) and it was found that this new immunosuppressant has a better and that it may be an immuno-suppressant of the new generation to come. This is achieved in the present invention.

In particular, the present invention provides a physiologically active kanglemycin C which is characterized by the following physicochemical properties, and salts thereof. 1. Relative molecular mass (EI-MS): m / z 326 (M +) 2. Molecular formula: θθ ^ 3. Melting point: 170 ° C (with decomposition) 4. Optical rotation: + 150 0 (C 0.57 , MeOH) 5. ultraviolet absorption spectrum (t 1 OH): (log £) 232 (4> 6 L)> 356 (3> 93) 6. infrared absorption spectrum (KBr) (cm -1): 3400, 1690, 1650, 1640 7. 13G-NMR (100 MHz, DMSO-d6): 204.02 (s), 160.58 (s), 70.92 (s), 20.47 (t), 195.0 (s), 159 , 50 (s), 136.66 (d), 134.58 (s), 43.62 (d), 23.75 (q), 123.95 (d), 46.52 (t), 31, 47 (t), 44.89 (d), 117.69 (s), 122.68 (d), 118.21 (d), 196.90 (s), 54.93 (d). 1 H NMR (400 MHz, DMSO-d 6) 5.73 (1H, brs), 1.98 (3H, s), 2.79 (1H, d, J = 18.54), 2.35 (1H, d, J = 18.54), 1.47 (2H, m), 1.98 (1H, m), 1.79 (1H, m), 1.93 (1H, t), 7.30 (1H, d ), 7.75 (1H, t), 7.55 (1H, d), 2.91 (1H, d), 3.37 (1H, d) - 3 -

A second aspect of the invention is to provide a process for preparing a physiologically active kanglemycin C and salts thereof, comprising culturing a microorganism belonging to the genus Nocardia that produces kanglemycin C and recovering physiologically active kang-lemycin C by fermentation of the culture medium. A third aspect of the invention is to provide a microorganism in the genus Nocardia producing kanglemycin C. A fourth aspect of the invention is to provide a pharmaceutical composition for immunosuppression comprising, as an active ingredient, a physiologically active kanglemycin C or a pharmaceutically acceptable salt thereof.

1 illustrates the ultra-violet absorption spectrum of kanglemycin C, FIG. 2 shows the infrared absorption spectrum (KBr) of kanglemycin C, FIG. Fig. 3 shows a &lt; 1 &gt; C-NMR spectrum (100 MHz, DMSO-d &lt; 8 &gt;) of kangemycin G and &lt; 4 &gt;-spectrum (400 MHz, DMSO-dg) of kanglemycin C in FIG.

The Kanglemycin C of the invention has a relative molecular weight of 326-&lt; &apos; &gt; K &lt; 0 &gt; K in physicochemical properties on the previous page, which is an extremely small comparison with the relative molecular weights of known immunosuppressants.

Optical rotation of kanglemycin C is: + 150 ° (C 0.57, MeOH)

Kanglemycin O demonstrates maximum absorption in methanol at 232 nma of 356 nm in the ultraviolet absorption spectrum; The absorption pattern at 3400 cm @ -190 cm, 1650 cm @ -1, 1640 cm @ -1, 750 cm @ -1, etc. is shown in the infrared absorption spectrum. The molecular formula (C19 H18 O5) has been analyzed by elemental analysis. The solvent is non-soluble in water and in a non-polar organic solvent, and the purified kanglemycin G forms yellow needles.

The physiologically active kanglemycin C of the present invention is obtained by culturing the microorganism belonging to the genus Koclemycin C producing Nocardia by producing and collecting kanglemycin C and recovering kanglemycin C from the fermentation broth thus obtained.

A representative example of a microorganism capable of producing kanglemycin C is Nocardia mediterranei var, kanglensis - 4 - 1747-64, belonging to the genus Nocardia and harvested in the People 's Republic of China. Bacteriological properties, physiological properties and others related to this strain are set forth below. 1. iiiiorfological properties

The strain was observed after incubation at 28 ° C for 2 days. The hyphae were simple with a marginal portion of corrugated or loop-like. Fragmentation of the bottom layer of my-celia substrate was observed. No sporangium or vortex is formed. The surface of the spore is smooth or rough and sometimes thorny. The spores are cylindrical and have a magnitude of 0.6 (µm - 0.8 µm x 0.8 µm - 1.3 £ un. The dispute consists of about de-hundred strings.

The growth stage at 28 ° C observed in various media after two weeks is shown in Table 1

Table 1

Environment .. Growth Aerial mycelium Substrate mycelium Soluble pigment Sucrose-nitrate agar good mild, white to pale yellow-orange light yellow-pale orange brown Glucose-asparagine agar mild mild, white to pale yellow-orange pale yellow-pale yellowish yellowish Glycerin-asparagine agar good mild, white to light yellow-orange light yellow-yellow light-orange brown 3-star-inorganic salt agar mild mild, white pale yellow-pale brownish brownish Tyrosine agar mild mild, white pale yellowish pale yellowish brownish nutrient mild mild, white to pale yellow-orange pale yellow-orange no Kvasnico-malt agar mild mild, white light brown yellowish oatmeal Agar mild mild, pale orange to pale yellowish pale orange yellowish Pepton-yeast © iron agar mild light yellow none f - 5 - 3 · Physiological properties

Optimum Growth Temperature: 27 ° C to 37 ° Nitrate Reduction: Negative

Liquefaction of gelatin (glucose-peptone-gelatin medium, 20 ° C): negative

Hydrolysis of starch (starch-inorganic salt agar): negative

Collected milk coagulation: positivePeptonization of collected milk: positive melanoid pigment formation: negative 4. Used carbon sources (Pridham-Gottlieb agar medium) L-Arabinose + D-Xylose + D-Glucose + D-Fructose + Sucrose + Inositol + L-Rhamnose + Rafinose - D-Mannitol + 5. Diaminopimelic acid in the cell wall Meso-diaminopimelic acid

In short, the cell wall of this strain is meso-diaminopimelic acid. According to the International Strepto-myces Project (abbreviated to I3P), hyphae forming controversies belong to the section:

Rectiflexibles or Recticuliaperti; the surface of the spore is smooth or rough; the color of the adult hyphae is from a series of white colors; the strain does not form melanaid pigments or other pigment in the environment; the color of the substrate mycelium is light brown or pale yellow, a division is observed; the chain grows using carbon sources such as L-arabinose, D-glucose, D-fructose, sucrose, inositol, L-rhamnose, D-xylose and D-mannitol, but does not grow with rafinose.

Based on the foregoing properties, a bacteriological study was conducted according to the Bergeys kanual of Determinative Bacteria, 8th edition (1974); the results showed that this strain is found in the genus Nocardia. Therefore, the strain was named Nocardia medi-terranei var, kanglensis 1747-64 (referred to as strain 1747-64) - 6 -

This strain was deposited in the Committee of Preservation and Control of Chinese Kicroorganism in the People's Republic of China on February 20, 1991 and is available under No. CGkCC No. This is also stored in the Permentation Research Institute of the Industrial Science &amp; Techology of Japan on January 28, 1992 under the Budapest Treaty and is available under the name No. FERK BP-3713.

The Kanglemycin C of the present invention can be prepared by culturing a microorganism, the calcining kanglemycin C, belonging to the genus Nocardia, and obtaining physiologically active kanglemycin C by fermentation of the broth.

Any medium can be used as an environment for culturing kang-lemycin G-producing bacteria, as long as it contains adequate sources of nutrients that kanglemycinC-producing bacteria can assimilate. Examples of carbon sources that can be used are glycerin, glucose, maltose, sucrose, dextrin, starch, olefin and fats, etc. As nitrogen sources that can be used are organic substances such as soy suspension, inoculated suspension of seeds, meat extract, peptone, dried yeast, yeast yeast suspension, yeast extract, dipped corn solution, etc. inorganic substances such as ammonium nitrate, ammonium sulfate, sodium nitrate, calcium carbonate, ammonium chloride, and the like.

If necessary and necessary, inorganic salts such as sodium chloride, potassium chloride, malate, heavy metal salts, etc., amino acids and vitamins can be added.

In order to prevent foaming during fermentation, it is also advisable to add conventional defoaming agents, such as silicone, soybean oil, etc., in a conventional manner.

The preferred method for culturing is deep air blasting, which is conventionally used in the manufacture of anti-biotics.

The cultivation temperature is not particularly limited as far as the okanglemycin is concerned, it can be produced, for example, at a temperature in the range of 25 ° C to 37 ° C, without kanglemycin C producing growth bacteria being particularly prevented. C to 32 ° C.

Cultivation generally lasts until sufficient accumulation of kanclemycin C occurs. The culture time varies depending on the culture medium composition and incubation temperature. Typically, the desired kanglemycin G is obtained by culturing for 20 hours to 60 hours. - 7 -

The cultivation can be carried out as described in more detail below in Example 1. Strain 1747-64 is subjected to oblique cultivation in an oblique cultivation medium composed of starch, sodium chloride, potassium nitrate, agar, and so on. mycelium cake (layer) and inoculate the seeded culture medium. The inoculated culture medium consists of glucose, yeast extract, soy extract, potassium hydrogen phosphate, ammonium sulfate, etc. Incubation takes place inoculated with culture medium at 25 ° C to 32 ° C for 24 hours to 55 hours. The soaked fermentation broth is transferred to a fermentation medium followed by culturing at 25 ° C to 32 ° C for 68 hours to 96 hours. The fermentation medium is composed of glucose, yeast extract, soybean suspension, peptone, calcium carbonate, etc. Kanglemycin C is harvested from the fermentation broth thus obtained. An appropriate appropriate isolation method is used to isolate kanglemycin C from the fermentation broth thus obtained.

For example, the fermentation broth is filtered and the mycelium isolated by known isolation methods, for example by centrifugation. From the supernatant, kanglemycin C can be isolated, purified, and extracted by solvent using a suitable solvent, chromatographically using adsorption or ion exchange capability, simply or in combination.

The Kanglemycin C of the present invention has the effect of strongly inhibiting immune cells, particularly the spleen cell conversion. According to the present invention, an immunosuppressant comprising kanglemycin C or a pharmaceutically acceptable salt thereof as active ingredient is prepared. The immunosuppressant of the present invention can be administered to warm-blooded animals, including humans, at an effective dose of kang-lemycin G. This immunosuppressant can be given parenterally by injection; alternatively, the immunosuppressant may also be administered orally or by other parenteral means than injection. The dose of kanglemycin C may be highly dependent on the subject being administered or the route of administration, but in general the dosage ranges from 0.1 mg / kg / day to 100 mg / kg / day, preferably 0.5 mg / kg / day. kg / day to 50 mg / kg / day.

The pharmaceutical compositions are not particularly limited.Kanglemycin C is prepared in the form of a liquid or solid prodrug containing from 0.01 wt. % to 50 wt. % C, based on the total weight of the preparation, together with the commonly used pharmaceutical additives, for example carriers, auxiliaries, etc. This preparation is administered orally or parenterally.

As described above, kanglemycin, a new physiologically active substance, can be obtained from the fermentation broth 1747-64 belonging to the genus Nocardia. Kanglemycin C strongly inhibits the transformation of immune cells, especially the spleen, and strongly inhibits tumor cell proliferation . In addition, kangemycin C may advantageously be prepared from an industrial perspective because it has an extremely low molecular weight relative to known immunosuppressants.

It is believed that kanglemycin C is a promising next generation immunisuppressant since kanglemycin C eliminates various non-benefits associated with known immunosuppressants. The invention is further described in the following Examples and Test Example. EXAMPLES Example 1

Canglemycin C production

Strain 1747-64 is cultured and fermented. The fermentation broth thus obtained is extracted with an organic solvent. Kanglemycin C is isolated and purified from the extract by column chromatography. Conditions are detailed: 1. oblique culture medium (%): KNO 0.1; NaCl 0.5; OHPO ^ 5 0.05, FeSO ^ 0.01; starch 2; agar 1.7; cold boiled water, pH7.0; incubating at 28 ° C for 5-7 days; 2. Vaccinated culture medium (%): glucose 2.5; yeast powder0.5; KC1 0.25; soy suspension 0.5; K2HP0 ^ 0.02; (NH 4 · SO 4 · 0.5; MgSO 4 · 0.02; CaCO 3 · 0.5; drinking water 100 ml; (pH not adjusted) 3. Composition of fermentation medium (%): glucose 3; 1, soybean suspension 0.5, peptone 0.2, CaCO 3 0.1, drinking water 100 ml (pH not adjusted) The media described above are all sterilized at 121 ° C for 30 minutes in an autoclave and stored for use.

The 1747-64 culture loop was inoculated into a 250 ml shaker flask filled with 50 ml grafted culture medium and cultured at 28 ° C for 48 hours in a rotary shaker (130-200 rpm). The soaked culture broth is transferred to a 500 ml shake flask containing 50 ml of the fermentation broth in a 10 &amp;amp;amp;amp; (by volume). After cultivation at 28 ° C for 72 hours in a rotary shaker (at 180-200 rpm), a fermentation broth is obtained.

The fermentation broth thus obtained is filtered and the pH of the filtrate is adjusted to 4 by the addition of inorganic acid. The filtrate is extracted with butyl acetate. The extract was concentrated and the obtained concentrate was subjected to silica gel chromatography. After elution with chloroform-methanol (30: 1), the active fractions in the eluate were combined and concentrated. The concentrate thus obtained is further purified on a Sephadex LH-20 ethanol column. The active fractions of the ethanol eluate are combined and concentrated. Recrystallization from ethanol gives purified kanglemycin C. Kanglemycin Gels to form yellowish needles and decomposes at 170 ° C. The relative molecular weight of this material is 326 and its optical activity is: E < k &gt; 2p + 150 [deg.] (C 0.57, MeOH); the maximum absorption spectrum in the ultraviolet region in methanol at 232 nm and 256 nm (see FIG. 1); in an infrared absorption spectrum at 3400 cm @ -1 '3', 1690 cm @ 1650 cm @ 3, 3 @ 64O cm @ 3, 750 cm @ 3 (see FIG. 2). This compound is soluble in a polar organic solvent but insoluble in water and in non-polar organic solvents

Isolation and purification of kanglemycin G To 20 liters of fermentation broth obtained in a similar manner to Example 1, 20 liters of butyl acetate were added. The pH of this mixture was adjusted to 4.0 with 3K-HCl and extracted. The organic phase is dried to dryness under reduced pressure to give 3.12 g of residue. This residue was dissolved in methanol and 30 g of silica gel (manufactured by Chingtao Kako Factor) -methanol was added to the solution to dissolve the residue (30 ml). 10 - evaporate to dryness under reduced pressure. The residue was placed on a S00 mL column with silica gel and chloroform. The column was developed with chloroform: methanol (30: 1) and the desired fractions were collected. This gives 25.3 mg of a brownish powder. This powder is dissolved in ethanol. The solution is then passed through a column with 300 ml of Oephadex Li-20 (Trade Mark; manufactured in Pharmacia Fine Chemicals Inc) and developed with ethanol. The desired fractions are collected and evaporated to dryness under reduced pressure. The residue is recrystallized from a small amount of ethanol to give 9.5 mg of kanglemycin C as yellowish needles.

Melting point: 170 ° C (decomposition) ΞΙ-MS m / z: 326 (M +)

Elemental Analysis:

Calculated: C 69.93; H 5.55 Found: C 69.5δ; H 5.60 13 C-NMR (100 MHz, DMSO-d 6) (see Figure 3): 204.02 (s); 160.58 (s), 70.92 (s); 20.47 (t); 195.0 (s); 159.50 (s); 136.66 (d), 134.58 (a); 43.62 (d); 23.75 (q), 123.95 (d), 46.52 (t); 31.47 (t); 44.89 (d); 117.69 (s); 122.68 (d); 118.21 (d); 196.90 (s); 54.93 (d) 1 H-NMR (400 MHz, D 6 SO-dg) 5.78 (1H, brs); 1.98 (3H, s); 2.79 (1H, d, J = 18.54); 2.35 (1H, d, J = 18.54); 1.57 (2 H, m); 1.98 (1 H, m); 1.79 (1 H, m); 1.98 (1 H, t); 7.30 (1H, d), 7.75 (1H, t); 7.55 (1H, d), 2.91 (1 H, d); 3.37 (1H, d) HPLC: Column: Senshu Pak ODS-2151-A 6x5 x 150 mm Temperature: 35 ° CDetection: UV (232 nm)

Mobile phase: CH 2 CN: Ο, ΙΜ-phosphate buffer (pH 6.0) = 4: 6

Retention time: 7.41 minutes (kanglemycin C) 11 -

Testing example

Immunosuppressive efficacy and tumor cell growth inhibitory activity of kanglemycin C and toxicity At 0.5 µg / ml kanglemycin G, the mouse spleen lymphocyte conversion rate in mice reached 96.9 When spleen cells were cultured in mice did not show kanglemycin C toxicity up to a dose of 40 µg / ml, indicating that no toxicity to spermatogenic cells at concentrations up to 1 mg / ml was observed. No death was observed when kanglemycin C was administered intravenously to mice at 2 mg / kg. This confirms that this toxicity is very low. kanglemycin C inhibits K562 leukemia cell proliferation at a concentration of 0.1 µg / ml or less and inhibits HeLa cell proliferation at 0.1 µg / ml. Example 3 Preparation of tablets

A mixture of 30 parts by weight of kanglemycin C, 120 parts by weight of crystalline lactose, 147 parts by weight of crystalline cellulose and 3 parts by weight of magnesium stearate is tabletted using a V-shaped mixer to give tablets of 300 mg.

Claims (9)

12-26 (M) PATENT N A R ϋ K Ϊ 1. Molecular weight (SI-MS): m / z 3 Kanglemycin C, which is a physiologically active substance having the following physicochemical properties and its pharmaceutically acceptable salts. 2. A process for the preparation of kanglemycin C according to claim 1 which is a physiologically active substance and its salts, characterized in that the microorganism-producing kanglemycin C belonging to the genus Nocardia is cultured and kanglemycin C is obtained from the fermentation broth. 2. Molecular formula: J. Melting point: 170 ° C (decomposition) 3. A pharmaceutical composition according to claim 1, wherein the active ingredient is kanglemycin, which is a physiologically active substance or a pharmaceutically acceptable salt thereof. "ugd ΛΛ3Γ90V ΑΖ3ΊγΝλΛ avgn OSd Z 6 III '0 ε (π) T d, J = 18,54) L, 98 (1H, m); 4. The pharmaceutical composition of item 3, which is suitable for immunosuppression. 4. Optical activity: &amp; t] 2D + 150 ° (c ° '57 'ke0H) 5. A strain belonging to the genus Nocardia and capable of producing kang-lemycin C, which is a physiologically active substance. 5. Ultraviolet Absorption Spectrum (MeOH): (log £) 232 (4.61), 356 (3.93) 6. A strain capable of producing kanglemycin G according to point 3, which is Nocardia copperiterranei var, kanglensis 1747-64 (FERM BF-3718) ^ 6. Infrared absorption spectrum (KBr) (cm -1) 3400, 1690, 1650, 1640 7. 13 C-WR (100 MHz, DMSO-d 6): 204.02 (s); 160.58 (s); 70.92 (s); 20.47 (t); 195.0 (s); 159.50 (s); 136.66 (d); 134.58 (s); 43.62 (d); 23.75 (q); 123.95 (d); 46.52 (t); 31.4744.89 Cd); 117.69 (s); 122.68 (d); 118.21 (d); 196.90 (s); 54.93 (d). Δ-δ (400 MHz, DMSO-d 6) 5.78 (1H, brs); 1.98 (3H, s); 2.79 (1H, 2.35 (1H, d, J = 18.54), 1.47 (2H, m), 1.79 (1H, m), 1.98 (1H, t), 7.30 ( 1H, d), 7.75 (1H, t), 7.55 (1H, d), 2.91 (1H, d), 3.37 (1H, d). 7. A method for immunopressing comprising administering to a mammal a kanglemycin C of item 1 which is a physiologically active agent, or a pharmaceutically acceptable salt thereof, in a pharmacologically effective amount. Use of kanglemycin C, which is a physiologically active substance, or a pharmaceutically acceptable salt thereof, as a pharmaceutically active ingredient. Use of kanglemycin G, which is a physiologically active substance, or a pharmaceutically acceptable salt thereof, for the preparation of a pharmaceutical composition for immunosuppression.